The proposed research project aims at elucidating the molecular structures of adult and fetal red cell phosphofructokinase (PFK) proteins in terms of the types of monomeric subunits (whether similar or dissimilar) composing these tetrameric enzymes. Knowledge of the PFK subunit structures can directly provide a genetically defined molecular basis to explain a. the heterogeneity of clinical syndromes observed in PFK deficiency states; b. the lower content of PFK in newborn red cells. Both of these have been explained by postulating isozymes of PFK under separate genetic control and with different biochemical characteristics. This goal will be approached by the following means: 1. direct isolation and purification of various human isozymes; 2. characterization of their physico-chemical, kinetic, and regulatory properties; 3. elucidation of their subunit structures utilizing techniques of molecular hybridization and dissociation-reassociation experiments; 4. demonstration of structural-genetic identity or non-identity between isozymes using their immunochemical specificities. The preliminary data obtained in this laboratory have demonstrated that a. human muscle and liver isozymes are homotetramers, each composed of identical subunits (M4 and L4 respectively); b. adult red cell PFK is a heterogeneous mixture of three hybrid heterotetramers (M3L, M2L2, and ML3); c. together these five comprise a chromatographically and kinetically identifiable family of isozymes; d. newborn red cell PFK contains L4 in addition to three hybrid isozymes, and this isozyme is probably responsible for the observed low enzyme activity of newborn red cells. These initial observations need not only confirmation and some clarifications, but also some extended studies to yield more complete and meaningful data. The biochemical techniques developed during this research project could be used as a model to investigate the molecular structures of other key red cell enzymes, such as, for instance, pyruvate kinase isozyme(s).